Laura Zagato

Association of the α-Adducin Locus With Essential Hypertension

Abstract Previous studies on genetic rat hypertension have shown that polymorphism within the α-adducin gene may regulate blood pressure. Adducin is a cytoskeletal protein that may be involved in cellular signal transduction and interacts with other membrane-skeleton proteins that affect ion transport across the cell membrane. There is a high homology between rat and human adducin and pathophysiological similarities between the Milan hypertensive rat strain and a subgroup of patients with essential hypertension. Thus, we designed a case-control study to test the possible association between the α-adducin locus and hypertension. One hundred ninety primary hypertensive patients were compared with 126 control subjects. All subjects were white and unrelated. Four multiallelic markers surrounding the α-adducin locus located in 4p16.3 were selected: D4S125 and D4S95 mapping at 680 and 20 kb centromeric, and D4S43 and D4S228/E24 mapping at 660 and 2500 kb telomeric. Alleles for each marker were pooled into groups. Comparisons between control subjects and hypertensive patients were carried out by testing the allele-disease association relative to the marker genotype. The maximal association occurred for D4S95 (χ12 13.33), which maps closest to α-adducin. These data suggest that a polymorphism within the α-adducin gene may affect blood pressure in humans.

Genomewide association study using a high-density single nucleotide polymorphism array and case-control design identifies a novel essential hypertension susceptibility locus in the promoter region of endothelial NO synthase

Essential hypertension is a multifactorial disorder and is the main risk factor for renal and cardiovascular complications. The research on the genetics of hypertension has been frustrated by the small predictive value of the discovered genetic variants. The HYPERGENES Project investigated associations between genetic variants and essential hypertension pursuing a 2-stage study by recruiting cases and controls from extensively characterized cohorts recruited over many years in different European regions. The discovery phase consisted of 1865 cases and 1750 controls genotyped with 1M Illumina array. Best hits were followed up in a validation panel of 1385 cases and 1246 controls that were genotyped with a custom array of 14 055 markers. We identified a new hypertension susceptibility locus (rs3918226) in the promoter region of the endothelial NO synthase gene (odds ratio: 1.54 [95% CI: 1.37–1.73]; combined P=2.58 · 10−13). A meta-analysis, using other in silico/de novo genotyping data for a total of 21 714 subjects, resulted in an overall odds ratio of 1.34 (95% CI: 1.25–1.44; P=1.032 · 10−14). The quantitative analysis on a population-based sample revealed an effect size of 1.91 (95% CI: 0.16–3.66) for systolic and 1.40 (95% CI: 0.25–2.55) for diastolic blood pressure. We identified in silico a potential binding site for ETS transcription factors directly next to rs3918226, suggesting a potential modulation of endothelial NO synthase expression. Biological evidence links endothelial NO synthase with hypertension, because it is a critical mediator of cardiovascular homeostasis and blood pressure control via vascular tone regulation. This finding supports the hypothesis that there may be a causal genetic variation at this locus.

Physiological Interaction Between α-Adducin and WNK1-NEDD4L Pathways on Sodium-Related Blood Pressure Regulation

The kidney plays an important role in salt and blood pressure (BP) homeostasis. In previous studies, variants in the genes for &agr;-adducin (ADD1), WNK1, and NEDD4L, which all regulate renal sodium absorption, have been associated with increased BP. However, findings have been inconsistent. We tested whether this is because of physiological interactions between the effects of variants in these genes. We assessed the single and combined effects of the ADD1 (Gly460Trp), WNK1 (rs880054 A/G), and NEDD4L (rs4149601 G/A) polymorphisms on renal and BP response to an acute Na load (n=344 subjects), BP decrease after 1 month of treatment with 12.5 mg of hydrochlorothiazide (n=193), and ambulatory 24-hour BP (n=690). Individually, the variants showed modest effects on some of the studied phenotypes. We found the ADD1 Trp allele to be permissive for the effects of variants of the other genes. In combination, the same variants (ADD1 Trp/WNK1 GG/Nedd4L GA+AA) showed a consistent effect on renal Na handling (P=0.009) and acute BP response to a saline infusion (P=0.021), BP lowering after thiazide treatment (P=0.008), and nocturnal systolic BP (P=0.044). Physiological interaction between the ADD1 and WNK1-NEDD4L pathways influences the effects of variants in these genes on sodium-related BP regulation. Relatively common alleles in the ADD1, WNK1, and NEDD4L genes when present in combination may have significant effects on renal sodium handling, BP, and antihypertensive response to thiazides.

Angiotensin-Converting Enzyme I/D and α-Adducin Gly460Trp Polymorphisms: From Angiotensin-Converting Enzyme Activity to Cardiovascular Outcome

The angiotensin-converting enzyme (ACE) I/D and the &agr;-adducin (ADD1) Gly460Trp polymorphisms are associated with cardiovascular risk factors. In a prospective population study and in cell models, we investigated the combined effects of these 2 polymorphisms. We randomly recruited 1287 white subjects (women: 50.0%; mean age: 55.9 years). We obtained outcomes from registries and repeat examinations (median 3). Over 9.0 years (median), 178 fatal or nonfatal cardiovascular events occurred. In ADD1 Trp allele carriers, the multivariate-adjusted hazard ratios associated with ACE DD versus I were 1.72 (P=0.007) for total mortality, 2.35 (P=0.02) for cardiovascular mortality, 2.02 (P=0.005) for all cardiovascular events, and 2.59 (P=0.03) for heart failure. In contrast, these hazard ratios did not reach significance in ADD1 GlyGly homozygotes (0.08≤P≤0.90). The positive predictive value and attributable risk associated with ACE DD homozygosity combined with mutated ADD1 were 36.2% and 10.3%, respectively. To clarify our epidemiological observations, we investigated the effects of mutated human ADD1 on the membrane-bound ACE activity in fibroblasts from 51 volunteers and in transfected human embryonic kidney cells (31 experiments). In fibroblasts (5.10 versus 3.63 nanomoles of generated hippuric acid per milligram of protein per minute; P=0.0021) and human embryonic kidney cells (1.086 versus 0.081 nmol/mg per minute; P=0.017), the membrane-bound ACE activity increased in the presence but not absence of the ADD1 Trp allele. In conclusion, the combination of ACE DD homozygosity and mutated ADD1 worsened cardiovascular prognosis to a similar extent as classic risk factors, possibly because of increased membrane-bound ACE activity in subjects carrying the ADD1 Trp allele.

Role of the adducin family genes in human essential hypertension

Objective In both humans and rats, polymorphisms of the alpha adducin (ADD1) gene are involved in renal sodium handling, essential hypertension and some of its organ complications. Adducin functions within cells as a heterodimer composed of various combinations of three subunits that are coded by three genes (ADD1, 2, 3) each located on a different chromosome. Design These characteristics provide the biochemical basis for investigating epistatic interactions among these loci. Methods We examined the three adducin gene polymorphisms and their association with ambulatory blood pressure (ABPM) and with plasma levels of renin activity (PRA), endogenous ouabain (EO), in 512 newly discovered and never-treated hypertensive patients. Results Relative to carriers of the wild type (Gly/Gly) ADD1 gene, patients carrying the mutated Trp ADD1 allele had higher blood pressure (systolic blood pressure (SBP) 143.2 ± 1.0 versus 140.6 ± 0.6 mmHg P = 0.027 and diastolic blood pressure (DBP) 94.2 ± 0.77 versus 92.3 ± 0.5 mmHg, P = 0.03), lower PRA and EO, consistent with the hypothesis of the renal sodium retaining effect of the Trp allele. Polymorphisms in the ADD2 and ADD3 genes taken alone were not associated with these variables. However, the differences in SBP and DBP between the two ADD1 genotypes were greatest in carriers of the ADD3 G allele (around + 8 mmHg). The significance of the interaction between ADD1 and ADD3 ranged between P = 0.020 to P = 0.006 according to the genetic model applied. Conclusions The interaction of ADD1 and ADD3 gene variants in humans is statistically associated with variation in blood pressure, suggesting the presence of epistatic effects among these loci.

Genes involved in vasoconstriction and vasodilation system affect salt-sensitive hypertension.

The importance of excess salt intake in the pathogenesis of hypertension is widely recognized. Blood pressure is controlled primarily by salt and water balance because of the infinite gain property of the kidney to rapidly eliminate excess fluid and salt. Up to fifty percent of patients with essential hypertension are salt-sensitive, as manifested by a rise in blood pressure with salt loading. We conducted a two-stage genetic analysis in hypertensive patients very accurately phenotyped for their salt-sensitivity. All newly discovered never treated before, essential hypertensives underwent an acute salt load to monitor the simultaneous changes in blood pressure and renal sodium excretion. The first stage consisted in an association analysis of genotyping data derived from genome-wide array on 329 subjects. Principal Component Analysis demonstrated that this population was homogenous. Among the strongest results, we detected a cluster of SNPs located in the first introns of PRKG1 gene (rs7897633, p = 2.34E-05) associated with variation in diastolic blood pressure after acute salt load. We further focused on two genetic loci, SLC24A3 and SLC8A1 (plasma membrane sodium/calcium exchange proteins, NCKX3 and NCX1, respectively) with a functional relationship with the previous gene and associated to variations in systolic blood pressure (the imputed rs3790261, p = 4.55E-06; and rs434082, p = 4.7E-03). In stage 2, we characterized 159 more patients for the SNPs in PRKG1, SLC24A3 and SLC8A1. Combined analysis showed an epistatic interaction of SNPs in SLC24A3 and SLC8A1 on the pressure-natriuresis (p interaction = 1.55E-04, p model = 3.35E-05), supporting their pathophysiological link in cellular calcium homeostasis. In conclusions, these findings point to a clear association between body sodium-blood pressure relations and molecules modulating the contractile state of vascular cells through an increase in cytoplasmic calcium concentration.

Genetic Mapping of Blood Pressure Quantitative Trait Loci in Milan Hypertensive Rats

In a previous study, by using a candidate gene approach, we detected in both Milan hypertensive rats and humans a polymorphism in the &agr;-adducin gene (ADD1) that was associated with blood pressure and renal sodium handling. In the present study, a genomewide search with 264 informative markers was undertaken in 251 (Milan hypertensive strain × Milan normotensive strain) F2 rats to further investigate the contribution of the adducin gene family (Add1, Add2, and Add3) and to identify novel quantitative trait loci (QTLs) that affect blood pressure. The influence of 2 different methods of blood pressure measurement, the intracarotid catheter and the tail-cuff method, was also evaluated. We found evidence that QTLs affected systolic blood pressure (SBP) measured at the carotid (direct SBP) on rat chromosome 1 with a logarithm of the odds (LOD) score peak of 3.3 on D1Rat121 and on rat chromosome 14 on Add1 locus (LOD=3.2). A QTL for SBP measured at the tail (indirect SBP) was found on rat chromosome 10 around D10Rat33 (LOD=5.0). All of these QTLs identified chromosomal regions not detected in other rat studies and harbor genes (Na+/H+ exchanger A3; &agr;-adducin; &agr;1B-adrenergic receptor) that may be involved in blood pressure regulation. Therefore, these findings may be relevant to human hypertension, also in consideration of the biochemical and pathophysiological similarities between MHS and a subgroup of patients of primary hypertension, which led to the identification of &agr;-adducin as a candidate gene in both species.

Blood pressure in relation to three candidate genes in a Chinese population.

Objective In a prospective analysis of a Caucasian population, we recently found that the genes encoding angiotensin-converting enzyme (ACE, I/D polymorphism), α-adducin (Gly460Trp) and aldosterone synthase (–344C/T) jointly influence the incidence of hypertension. We therefore investigated the association between blood pressure and these three genes in a Chinese population. Methods We genotyped 479 Han Chinese from 125 nuclear families recruited in northern China via random sampling (∼75%) and at specialized hypertension clinics (∼25%). We performed population-based and family-based association analyses using generalized estimating equations (GEE) and the quantitative transmission disequilibrium test (QTDT), respectively, while controlling for covariables. Results The participants included 239 (49.9%) women and 132 (27.6%) hypertensive patients, of whom 77 took antihypertensive drugs. The blood pressure, measured at the subjects’ homes, averaged 126/80 mmHg. Mean values of urinary sodium, potassium and Na+/K+ ratio were 226 mmol/day, 37 mmol/day and 6.31, respectively. In adjusted GEE analyses, systolic blood pressure was 9.3 mmHg (95% confidence interval 3.6–15.0 mmHg; P = 0.001) and 14.6 mmHg (95% confidence interval 3.4–25.8 mmHg; P = 0.01) higher in the ACE DD than II subjects among the α-adducin TrpTrp (n = 141) and aldosterone synthase CC (n = 33) homozygotes, respectively (P ⩽ 0.05 for interactions of the ACE genotype with the α-adducin and aldosterone synthase polymorphisms). Among 40 informative offspring homozygous for the α-adducin Trp allele, systolic blood pressure was significantly associated with transmission of the ACE D allele (β = 5.5 mmHg; P = 0.046). Conclusions The ACE I/D, α-adducin Gly460Trp and aldosterone synthase –344C/T polymorphisms interact to influence systolic blood pressure in Chinese, suggesting that these genes might indeed predispose to hypertension, especially in an ecogenetic context characterized by a high salt intake.

Cardiovascular Risk in Relation to α-Adducin Gly460Trp Polymorphism and Systolic Pressure. A Prospective Population Study

Preliminary evidence from 1 case-control study suggested that in hypertensive patients, the α-adducin 460Trp allele might be associated with a 2-fold higher risk of coronary heart disease. In a prospective population study, we investigated whether the α-adducin Gly460Trp polymorphism predicted mortality and morbidity. From August 1985 until July 2003, we randomly recruited 2235 Belgian residents. We obtained information on vital status (until July 1, 2004) and the incidence of events via registries and repeat examinations (median 3). In Cox regression, before and after adjustment for other risk factors, we found strong interaction between systolic blood pressure at baseline, analyzed as a continuous variable, and the α-adducin polymorphism in relation to total (P=0.01) and cardiovascular mortality (P=0.007) and all cardiovascular (P=0.003), cardiac (P=0.001), and coronary events (P=0.03). The hazard ratio for total mortality associated with the Trp allele relative to GlyGly homozygosity was 2.30 (95% confidence interval, 1.12 to 4.72; P=0.02) in patients with stage-2 systolic hypertension (≥160 mm Hg) and 0.88 (0.61 to 1.26; P=0.48) in the other participants. For all cardiovascular complications, these estimates were 2.94 (1.28 to 6.74; P=0.01) and 0.83 (0.58 to 1.20; P=0.32), respectively. For all cardiovascular events, the positive predictive value and the attributable risk associated with the Trp allele in patients with stage-2 systolic hypertension were 76.9% and 44.3%, respectively. In conclusion, the α-adducin Gly460Trp polymorphism, in combination with systolic blood pressure, is a strong predictor of cardiovascular mortality and morbidity.

Gly460Trp α-adducin mutation as a possible mechanism leading to endolymphatic hydrops in Ménière's syndrome

Objective: Ménières disease (MD) is an inner ear disorder characterized by recurrent episodic vertigo, hearing loss that is fluctuating in the first stages, aural fullness, and tinnitus. Raised endolymphatic pressure (hydrops) is commonly accepted as a causal condition. Approximately 90% of cases of MD are sporadic, whereas the remaining 10% of cases are linked to genetic factors. The ionic composition of endolymph may also depend on the activity of Na+, K+-ATPase. Adducin is a heterodimeric cytoskeleton protein consisting of 3 subunits (&agr;, &bgr;, and &ggr;) coded by 3 different genes (ADD1, ADD2, and ADD3). ADD1 Gly460Trp polymorphism is associated with salt-sensitive hypertension and increased Na+-K+ pump activity in transfected cells. This study aims to verify the role of adducin in the development of MD. Methods: We genotyped 28 patients affected by definite MD according to American Academy of Otolaryngology-Head and Neck Surgery Foundation criteria. Results were compared with those from 2 different control populations (normotensive control group from San Raffaele Hospital and general population group). Results: We have not found any significant difference in the distribution of ADD2 C1797T and ADD3 IVS11+386A/G polymorphism genotypes. On the other hand, the frequency of ADD1 Trp allele is significantly increased in patients with MD compared with controls. Conclusion: We present data supporting the possibility that increased Na+, K+-ATPase activity may be one of the pathologic mechanisms inducing hyperosmolarity in endolymph which, in turn, may lead to hydrops.